I. Nasal Delivery Systems
Until recently, antibiotics, anti-inflammatory steroids and decongestants have been administered intranasally only for their local action, e.g., nasal decongestion and bronchodilation. The observation that systemic side-effects appeared in some cases led to the conclusion that the nasal mucosa permits the systemic availability of some drugs. Nasal delivery offers promising alternative to parenteral administration of therapeutic agents that cannot tolerate the rigorous gastrointestinal environment after oral administration. Nasal administration may therefore be considered as one of the possible alternatives to delivering peptides and protein drugs.
The primary function of the nose is olfaction, but it also filters airborne particulates, as well as heat and humidified inspired air. In adult humans, the nasal cavities are covered by a 2.0 to 4.0 mm thick mucosa (Mygind, Nasal Allergy, Blackwell Scientific, Oxford (1979)). The volume of the nasal cavity is about 20 ml, and its total surface area is about 180 cm.sup.2 (Schreider, Toxicology of the Nasal Passage, Hemisphere, Washington, D.C., pages 1-23 (1986)). Absorption of therapeutic agents across the nasal mucosa results in direct systemic exposure, thus avoiding the first-pass hepatic metabolism associated with oral administration. However, an alternative first-pass effect is created by the metabolic activity within the nasal mucosa (Sarkar, Pharmacol. Res., 9:1-9 (1992)).
Although the bioavailability of peptides and proteins from the nasal mucosa is substantially improved over the oral route, it is still far from optimal when compared to the intravenous route. This limitation may be attributed to the resistance encountered by macromolecules in penetrating the nasal mucosa through the paracellular pathway (Sackar, supra).
Studies on the use of the paracellular pathway have not been extensively explored, mainly because of lack of information on tight junctions (tj) structure and function. That is, entry of molecules through the paracellular pathway is primarily restricted by the tj (Gumbiner, Am. J. Physiol., 253:C749-C758 (1987); and Madara, J. Clin. Invest., 83:1089-1094 (1989)).
In transmission electron microscopy, tj appear as an approximately 80 nm long region at the boundary of neighboring cells in which the plasma membranes of adjacent cells are brought into close opposition (Farquhar et al, J. Cell Biol., 17:375-412 (1963)). This structure circumscribes epithelial cells immediately below the apical domain, forming a seal between epithelial cells and their neighbors. This seal restricts diffusion of small molecules in a charge specific manner (Pappenheimer et al, J. Membrane Biol., 102:2125-2136 (1986) ; Madara et al, J. Cell Biol., 102:2125-2136 (1986); Claude et al, J. Cell Biol., 58:390-400 (1973); and Bakker et al, J. Membrane Biol., 11:25-35 (1989)), and completely occludes molecules with molecular radii larger then 11 .ANG. (Madara et al, J. Cell Biol., 98:1209-1221 (1985)). Thus, considerable attention has been directed to finding ways to increase paracellular transport by "loosening" tj.
To overcome the poor uptake from the nasal mucosa, absorption enhancers are employed in attempts to increase the extent of peptide absorption. Examples of these enhancers include bile salts (Duchateau et al, Int. J. Pharm. 31:193-196 (1986)), chelating agents (Lee, In: Delivery Systems for Peptide Drugs, Plenum, New York, pages 87-104 (1986)), and surfactants (Hirai et al, Int. J. Pharm. 9:165-169 (1981)). Since the penetration enhancers listed above promote peptide and protein absorption by perturbing membrane integrity, it is inevitable that varying extents of insult will occur to the mucosal tissues that are in contact with the enhancer (Lee, supra). The alteration of the membrane integrity can permanently damage the nasal membrane (Hirai et al, supra) and, consequently, makes the use of these substances unacceptable for chronic treatments in humans.
Thus, there has been a desire in the art to develop nasal absorption enhancers which do not have the above-discussed limitations.
II. Function and Regulation of Tight Junctions
The tj or zonula occludens (hereinafter "ZO") are one of the hallmarks of absorptive and secretory epithelia (Madara, J. Clin. Invest., 83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990). As a barrier between apical and basolateral compartments, they selectively regulate the passive diffusion of ions and water-soluble solutes through the paracellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol. 22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond, Physiologist, 20:10-18 (1977)).
Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, since the resistances of cell plasma membranes are relatively high (Madara, supra). The ZO represents the major barrier in this paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on the number of transmembrane protein strands, and their complexity in the ZO, as observed by freeze-fracture electron microscopy (Madara et al, J. Cell Biol., 101:2124-2133 (1985)).
There is abundant evidence that ZO, once regarded as static structures, are in fact dynamic and readily adapt to a variety of developmental (Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel et al, Cold Spring Harbor Symp. Quant. Biol., 40:443-455 (1976); and Schneeberger et al, J. Cell Sci., 32:307-324 (1978)), physiological (Gilula et al, Dev. Biol., 50:142-168 (1976); Madara et al, J. Membr. Biol., 100:149-164 (1987); Mazariegos et al, J. Cell Biol., 98:1865-1877 (1984); and Sardet et al, J. Cell Biol., 80:96-117 (1979)), and pathological (Milks et al, J. Cell Biol., 103:2729-2738 (1986); Nash et al, Lab. Invest., 59:531-537 (1988); and Shasby et al, Am. J. Physiol., 255(Cell Physiol., 24):C781-C788 (1988)) circumstances. The regulatory mechanisms that underlie this adaptation are still not completely understood. However, it is clear that, in the presence of Ca.sup.2+, assembly of the ZO is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation and modulation of an organized network of ZO elements, the composition of which has been only partially characterized (Diamond, Physiologist, 20:10-18 (1977)). A candidate for the transmembrane protein strands, occludin, has been identified (Furuse et al, J. Membr. Biol., 87:141-150 (1985)).
Six proteins have been identified in a cytoplasmic submembranous plague underlying membrane contacts, but their function remains to be established (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer (Gumbiner et al, Proc. Natl. Acad. Sci., USA, 88:3460-3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3). Most immunoelectron microscopic studies have localized ZO-1 to precisely beneath membrane contacts (Stevenson et al, Molec. Cell Biochem., 83:129-145 (1988)). Two other proteins, cingulin (Citi et al, Nature (London), 333:272-275 (1988)) and the 7H6 antigen (Zhong et al, J. Cell Biol., 120:477-483 (1993)) are localized further from the membrane and have not yet been cloned. Rab 13, a small GTP binding protein has also recently been localized to the junction region (Zahraoui et al, J. Cell Biol., 124:101-115 (1994)). Other small GTP-binding proteins are known to regulate the cortical cytoskeleton, i.e., rho regulates actin-membrane attachment in focal contacts (Ridley et al, Cell, 70:389-399 (1992)), and rac regulates growth factor-induced membrane ruffling (Ridley et al, Cell, 70:401-410 (1992)). Based on the analogy with the known functions of plaque proteins in the better characterized cell junctions, focal contacts (Guan et al, Nature, 358:690-692 (1992)), and adherens junctions (Tsukita et al, J. Cell Biol., 123:1049-1053 (1993)), it has been hypothesize that tj-associated plague proteins are involved in transducing signals in both directions across the cell membrane, and in regulating links to the cortical actin cytoskeleton.
To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the ZO must be capable of rapid and coordinated responses that require the presence of a complex regulatory system. The precise characterization of the mechanisms involved in the assembly and regulation of the ZO is an area of current active investigation.
There is now a body of evidence that tj structural and functional linkages exist between the actin cytoskeleton and the tj complex of absorptive cells (Gumbiner et al, supra; Madara et al, supra; and Drenchahn et al, J. Cell Biol., 107:1037-1048 (1988)). The actin cytoskeleton is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actin-binding proteins. An example of how the state of phosphorylation of an actin-binding protein might regulate cytoskeletal linking to the cell plasma membrane is the myristoylated alanine-rich C kinase substrate (hereinafter "MARCKS") MARCKS is a specific protein kinase C (hereinafter "PKC") substrate that is associated with the cytoplasmic face of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages 438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks to the membrane actin. Thus, it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid (Hartwig et al, Nature, 356:618-622 (1992)). Activated PKC phosphorylates MARCKS, which is released from the membrane (Rosen et al, J. Exp. Med., 172:1211-1215 (1990); and Thelen et al, Nature, 351:320-322 (1991)). The actin linked to MARCKS is likely to be spatially separated from the membrane and be more plastic. When MARCKS is dephosphorylated, it returns to the membrane where it once again crosslinks actin (Hartwig et al, supra; and Thelen et al, supra), These data suggest that the F-actin network may be rearranged by a PKC-dependent phosphorylation process that involves actin-binding proteins (MARCKS being one of them).
A variety of intracellular mediators have been shown to alter tj function and/or structures. Tight junctions of amphibian gallbladder (Duffey et al, Nature, 204:451-452 (1981)), and both goldfish (Bakker et al, Am. J. Physiol., 246:G213-G217 (1984)) and flounder (Krasney et al, Fed. Proc., 42:1100 (1983)) intestine, display enhanced resistance to passive ion flow as intracellular cAMP is elevated. Also, exposure of amphibian gallbladder to Ca.sup.2+ ionophore appears to enhance tj resistance, and induce alterations in tj structure (Palant et al, Am. J. Physiol., 245:C203-C212 (1983)). Further, activation of PKC by phorbol esters increases paracellular permeability both in kidney (Ellis et al C. Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol. 32):F293-F300 (1992)), and intestinal (Stenson et al, C. Am. J. Physiol., 265(Gastrointest. Liver Physiol., 28):G955-G962 (1993)) epithelial cell lines.
III. Zonula Occludens Toxin
Most Vibrio cholerae vaccine candidates constructed by deleting the ctxA gene encoding cholera toxin (CT) are able to elicit high antibody responses, but more than one-half of the vaccinees still develop mild diarrhea (Levine et al, Infect. Immun., 56(1):161-167 (1988)). Given the magnitude of the diarrhea induced in the absence of CT, it was hypothesized that V. cholerae produce other enterotoxigenic factors, which are still present in strains deleted of the ctxA sequence (Levine et al, supra). As a result, a second toxin, zonula occludens toxin (hereinafter "ZOT") elaborated by V. cholerae and which contribute to the residual diarrhea, was discovered (Fasano et al, Proc. Nat. Acad. Sci., USA 8:5242-5246 (1991)). The zot gene is located immediately adjacent to the ctx genes. The high percent concurrence of the zot gene with the ctx genes among V. cholerae strains (Johnson et al, J. Clin. Microb., 31/3:732-733 (1993), and Karasawa et al, FEBS Microbiology Letters, 106:143-146 (1993)) suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea typical of cholera. Recently, the zot gene has also been identified in other enteric pathogens (Tschape, 2nd Asian-Pacific Symposium on Typhoid fever and other Salomellosis, 47(Abstr.) (1994)).
It has been previously found that, when tested on rabbit ileal mucosa, ZOT increases the intestinal permeability by modulating the structure of intercellular tj (Fasano et al, supra). It has been found that as a consequence of modification of the paracellular pathway, the intestinal mucosa becomes more permeable. It also was found that ZOT does not affect Na.sup.+ -glucose coupled active transport, is not cytotoxic, and fails to completely abolish the transepithelial resistance (Fasano et al, supra).
More recently, it has been found that ZOT is capable of reversibly opening tj in the intestinal mucosa, and thus ZOT, when co-administered with a therapeutic agent, is able to effect intestinal delivery of the therapeutic agent, when employed in an oral dosage composition for intestinal drug delivery (WO 96/37196; U.S. patent application Ser. No. 08/443,864, filed May 24, 1995; and U.S. patent application Ser. No. 08/598,852, filed Feb. 9, 1996; each of which is incorporated by reference herein in their entirety).
In the present invention, it has been demonstrated, for the first time, that ZOT, when co-administered with a therapeutic agent, is able to enhance nasal absorption of a therapeutic agent. This finding was unexpected for the following reasons:
(1) Vibrio cholera naturally infect the intestinal mucosa, not the nasal mucosa; PA1 (2) The effect of ZOT on the intestinal mucosa is not uniform, i.e., ZOT exhibits its permeablizing effect only on the small intestine, not on the large intestine; and PA1 (3) The regional effect of ZOT appears to be related to the distribution of its receptor within the intestine, i.e., the receptor is expressed only by mature cells on the tip of the villi in the jejunum and ileum. It is not present on the surface of colonocytes (Fiore et al, Gastroenterology, 110:A323 (1996)). Heretofore, it was not known whether the ZOT receptor was expressed on the surface of nasal mucosa. PA1 (A) a therapeutic agent; and PA1 (B) a nasal absorption enhancing effective amount of zonula occludens toxin. PA1 (A) a therapeutic agent; and PA1 (B) a nasal absorption enhancing effective amount of zonula occludens toxin .
Thus, there was no reasonable expectation that a Vibrio cholera toxin, e.g., ZOT, would have any effect on tj of the nasal epithelia.